Cytochrome P450cin (CYP176A), Isolation, Expression, and Characterization

Abstract

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450 cin , CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450 cin gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450 cin may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at ϳ2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450 cin , it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450 cin by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K D ‫؍‬ 0.7 M), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450 cin catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.The cytochrome P450s (P450s) 1 are a superfamily of oxidative hemoproteins (1, 2) that carry out an enormous variety of oxidative transformations. These range from simple alkene epoxidation and heteroatom oxidation all the way through oxygen insertion into unactivated C-H bonds and C-C bond cleavage (3). P450s are broadly categorized as either soluble, e.g. most bacterial P450s, or as membrane bound/microsomal monooxygenases, e.g. most eukaryotic P450s. All P450s, however, contain a prosthetic heme group that is ligated to the protein backbone by a cysteinyl sulfur coordinated to the heme iron. It is this heme-thiolate moiety that is responsible for much of the chemistry carried out by these enzymes. The typical reaction catalyzed by a P450 is given by Reaction...

The Critical Role of Substrate-Protein Hydrogen Bonding in the Control of Regioselective Hydroxylation in P450cin

“…Expression and Purification of the P450cin N242A MutantThe P450cin N242A mutant was expressed and purified as reported in the literature for wild type P450cin (16). This yielded ϳ90 nmol of purified protein/liter of original culture.…”

“…Construction of N242A Mutant-The N242A mutant was generated in two PCR steps using forward (CP-1, 5Ј-CCGAAT-TCCATATGACTGCGACAGTCGC-3Ј) and reverse (CP-2, 5Ј-CCGAATTCTAGAGGAGCTTGCTCATTCCG-3Ј) flanking primers (16) and forward (N242A-f, 5Ј-CTCGGCG-GCATCGACGCCACCGCACGCTTCCTC-3Ј) and reverse (N242A-r, 3Ј-CGCCGTAGCTGCGGTGGCGTGCGAAG-GAGTCG-5Ј) mutagenic primers, pCW-P450cin as a template, and Vent DNA polymerase. In the first step two separate reactions were carried out, one with CP-1 and N242A-r and one with CP-2 and N242A-f.…”

Efficient catalytic turnover of cytochrome P450cam is supported by a T252N mutation

“…CYP176A (P450 cin ) from Citrobacter braakii, an organism that can grow on 1,8-cineole as its sole source of carbon and energy [10], is exceptional in that it does not retain the conserved, catalytically important hydroxyl residue (Thr or Ser) in the distal I helix (Fig. 2).…”

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

“…Class III CYPs are to date bacterial in origin and use flavin mononucleotide (FMN) containing flavodoxin as the reduction partner, first found for CYP176A1 (P450 cin ) metabolizing cineol [49]. Class IV is also of microbial origin found in an acidothermophilic archaeon.…”